Aspergillus fumigatus profilin

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of  A. fumigatus  profilin, methods of screening for  A. fumigatus  profilin modulators using biologically active  A. fumigatus  profilin, and kits for screening for  A. fumigatus  profilin modulators.

FIELD OF THE INVENTION

The present invention relates to a gene involved in actin dynamics andphospholipid hydrolysis in the yeast Aspergillus fumigatus and moreparticularly to the identification, isolation and cloning of this gene.This invention also relates to a method of using this gene to screen forcompounds with antifungal activity.

BACKGROUND OF THE INVENTION

Profilin is a ubiquitous 12-15 kDa eukaryotic actin andphosphotidylinositol-4,-5-biophosphate-(PIP₂) binding protein. Macheskyand Pollard (1993) Trends Cell Biol. 3:381-5. In mammalian cells, theprotein functions by binding PIP₂ which inhibits the phospholipidhydrolysis by phospholipase C (PLC) in resting cells. Goldsmidt-Clermontet al. (1990) Science 247:1575-8. When cells divide following activationof tyrosine kinase receptors, PLC is phosphorylated and therebyactivated to hydrolyze PIP₂ even in the presence of profilin.Goldschmidt-Clermont et al. (1991) Science 251:1231-3. This hydrolysisreleases the known messengers inositol triphosphate (IP₃) anddiacylglycerol (DAG) and profilin. Profilin moves from the plasmamembrane to the cytosol, where it binds to actin to catalyze therearrangment of the cytoskeleton. Goldschmidt-Clermont and Jamey (1991)Cell 66:419-21.

Profilin catalyzes release of adenosine diphosphate (ADP) from monomericor globular (G-) actin. Mockrin et al. (1980) Biochem. 19:5359-62;Goldschmidt-Clermont et al. (1991) J. Cell Biol. 113:1081-9. Thisactivity dramatically increases G-actin's exchange of ADP for ATP, whichdeactivates actin for polymerization into microfilaments andfaciliatates reorganization of the cytoskeleton. Carlier (1989) Int.Rev. Cytol. 115:139-70. Profilin therefore represents an importantcomponent of the eukaryotic cell division cycle as well as, by virtue ofits interaction with the actin cytoskeleton, overall cellular integrity,vesicular transport, and cell polarity.

Evidence suggests that profilin is important in the growth cycle ofyeasts. Null mutation in the Saccharomyces cerevisiae profilin gene islethal in many S. cerevisiae strain backgrounds. Magdolen et al. (1988)Mol. Cell Biol. 8:5108-15. In other S. cerevisiae strains, nullmutations produce a strain with highly abnormal morphology and severelyretarded growth rates. Haarer et al. (1990) J. Cell Biol. 110:105-14.While a role in phospholipid hydrolysis has not been shown, PIP₂dependent translocation of profilin from the plasma membrane into thecytoplasm has been demonstrated. Ostrander et al. (1995) J. Biol. Chem.46:27045-50. S. cerevisiae profilin has also been shown to interact withthe RAS/adenylate cyclase pathway. Vojtek et al. (1991) Cell 66:497-505.

There is a compelling need to prevent and treat systemic fungalinfections, many of which are fatal if untreated. Indeed, the 1980s and1990s witnessed a steep rise in Candida and Aspergillus infections(Musial, C E, Cockerill III, F R, Roberts G D. (1988) Clin Microb Rev1(4):349-364; Saral R. (1991) Reviews of Infectious Dis 13:487-492).Similar rises in zygomycosis, cryptococcosis, histoplasmosis and fusariainfection have also been noted. The reasons for the rise in fungalinfections are several, but a key factor is the growing population ofimmuno-compromised individuals. This group includes patients with HIVdisease (AIDS), older patients, patients who have undergone invasivesurgery, transplant patients and burn victims.

As the population of immunosuppressed individuals increases, so do thenumbers and types of fungal infections noted in these patients. Althoughcandidiasis remains the most common fungal infection in immunosuppressedpatients, aspergillosis, zygomycosis, and other infections byfilamentous fungi are a major problem for an increasing number ofpatients (Georgiev, V. St. (1 998) Infectious Diseases inImmunocompromised Hosts, CRC Press, Boca Raton, Fla.; and Fauci, A S.(1998) Emer Infect Dis. The endemic mycoses, especially histoplasmosisand coccidiodomycosis, also constitute a risk for patients. Atparticular risk for such infections are those with AIDS, those havingundergone bone marrow or organ transplants, those receiving chemotherapyand those who have had debilitating illness, sever injury, prolongedhospitalization, or long-term treatment with antibacterial drugs (NIAIDfact sheet, 1996).

According to the CDC's National Nosocomial Surveillance System, the rateof hospital-related fungal infections nearly doubled between 1980 and1990. In 1997, an estimated 240,000 individuals showed clinical symptomsof endemic mycoses. With the current approaches to treatment (primarilyamphotericin B and the azoles) the mortality rate in patients withsystemic fungal infections ranges from 30-100%, depending on thepathogen.

The severity of fungal infections increases as the immune system becomesmore dysfunctional. Fungi are among the most ubiquitous pathogens seenin patients with AIDS; virtually all major fungal pathogens causedisease in HIV-positive patients The majority of untreated HIV-positivepatients experience at least one episode of fungal infection and manyfungal infections are AIDS-defining illnesses in HIV-infectedindividuals (Phillips P. (1999).

Therefore, there is a desperate need for new antifungal agents. Therecent development of high-throughput screens for the isolation of suchagents presents an opportunity for meeting this need. Profilin isamenable to such screening approaches and thus represents an importantnew target for antifungal drugs.

SUMMARY OF THE INVENTION

The present invention concerns an isolated nucleic acid moleculeencoding A. fumigatus profilin. Preferably, the A. fumigatus profilinhas a sequence that has greater than 70%, 80%, or 90% amino acidsequence identity to SEQ ID NO:2 as measured using a sequence comparisonalgorithm.

In one aspect, the invention provides an isolated nucleic acid sequenceencoding A. fumigatus profilin, wherein the profilin has a sequence thathas greater than 70%, 80%, or 90% amino acid sequence identity to SEQ IDNO:2 as measured using a sequence comparison algorithm. In oneembodiment, the protein further specifically binds to polyclonalantibodies raised against SEQ ID NO:2.

In one embodiment, the nucleic acid encodes A. fumigatus profilin, or afragment thereof. In another embodiment, the nucleic acid encodes SEQ IDNO:2. In another embodiment, the nucleic acid has a nucleotide sequenceof SEQ ID NO:1.

In one aspect, the nucleic acid comprises a sequence which encodes anamino acid sequence which has greater than 70% sequence identity withSEQ ID NO:2, preferably greater than 80%, more preferably greater than90%, more preferably greater than 95% or, in another embodiment, has 98to 100% sequence identity with SEQ ID NO:2.

In one embodiment, the nucleic acid comprises a sequence which hasgreater than 55 or 60% sequence identity with SEQ ID NO:1, preferablygreater than 70%, more preferably greater than 80%, more preferablygreater than 90 or 95% or, in another embodiment, has 98 to 100%sequence identity with SEQ ID NO:1. In another embodiment providedherein, the nucleic acid hybridizes under stringent conditions to anucleic acid having a sequence or complementary sequence of SEQ ID NO:1.

In another aspect, the invention provides an expression vectorcomprising a nucleic acid encoding A. fumigatus profilin, wherein theprotein has a sequence that has greater than 70, 80, or 90% amino acidsequence identity to SEQ ID NO:2 as measured using a sequence comparisonalgorithm. The invention further provides a host cell transfected withthe vector.

In another embodiment, the protein comprises an amino acid sequence ofSEQ ID NO:2. In one aspect, the protein provided herein comprises anamino acid sequence which has greater than 70% sequence identity withSEQ ID NO:2, preferably greater than 80%, more preferably greater than90%, more preferably greater than 95% or, in another embodiment, has 98to 100% sequence identity with SEQ ID NO:2.

The invention features a substantially purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:2 or a fragment thereof and moreparticularly, the ATP hydrolysis pocket or P-loop of the amino acidsequence of SEQ ID NO:2 or a fragment thereof.

Also provided are modulators of the target protein including agents forthe treatment of fungal disorders. The agents and compositions providedherein can be used in variety of applications which include theformulation of sprays, powders, and other compositions. Also providedherein are methods of treating fungal disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a nucleic acid sequence encoding A.fumigatus profilin (SEQ ID NO:1).

FIG. 2 shows the amino acid sequence of A. fumigatus profilin (SEQ IDNO:2).

FIG. 3 shows the results of representative pyrene actin assays for A.fumigatus profilin. In the absence of profilin, pyrene actin assemblesinto filaments as evidenced by an increase in fluorescence over time.The addition of 5.0 uM profilin inhibits spontaneous assembly, dampeningany fluorescence increase, resulting in a trace that isindistinguishable from the G-actin control (no salt). An inhibitor ofprofilin will result in an increase in relative fluorescence intensity.The x-axes shows cycle number (1 cycle/30 sec) and the y-axes reflectarbitrary fluorescence intensity.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“A. fumigatus profilin” refers to proteins or polypeptides present in A.fumigatus that are capable of binding actin and PIP₂ and has thesequence described below.

An “anti-A. fumigatus profilin” antibody is an antibody or antibodyfragment that specifically binds a polypeptide encoded by the A.fumigatus profilin gene, cDNA, or a subsequence thereof.

“Biologically active” target protein refers to a target protein that hasone or more of the target protein's biological activities, including,but not limited to ability to bind actin and PIP₂ and hydrolyze ATP.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains a target protein or a fragment thereof or nucleicacid encoding a target protein or a fragment thereof. A biologicalsample comprises at least one cell or cell extract.

“Control region” refers to a nucleotide sequence that regulatesexpression of a nucleic acid or any subunit thereof, including but notlimited to any promoter, silencer, enhacer, splice site, transcriptionalinitiation element, transcriptional termination signal, polyadenylationsignal, translational control element, translational start site,translational termination site, and message stability element. Suchcontrol regions may reside 5′ or 3′ to the coding region or in intronsinterrupting the coding region.

A “comparison window” includes reference to a segment of any one of thenumber of contiguous position selected from the group consisting of from25 to 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity methods of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988) and Altschul et al. Nucleic Acids Res. 25(17): 3389-3402(1997), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and BLAST in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis., or by manualalignment and visual inspection (see, e.g., Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignment. It can also plot a dendrogram showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J Mol. Evol35:351-360(1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). As a general rule, PileUp canalign up to 500 sequences, with any single sequence in the finalalignment restricted to a maximum length of 7,000 characters.

The multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two alignedsequences. This cluster can then be aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences canbe aligned by a simple extension of the pairwise alignment of twoindividual sequences. A series of such pairwise alignments that includesincreasingly dissimilar sequences and clusters of sequences at eachiteration produces the final alignment and a consensus sequence ofconserved positions.

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

“High stringency conditions” may be identified by those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.015 M sodium citrate/0.1% sodium dodecyl sulfate at50-68° C.; (2) employ during hybridization a denaturing agent such asformamide, for example, 50% (v/v) formamide with 0.1 % bovine serumalbumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/50mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C.

“High throughput screening” as used herein refers to an assay whichprovides for multiple candidate agents or samples to be screenedsimultaneously. As further described below, examples of such assays mayinclude the use of microtiter plates which are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the gene contained within theexpression vector. Host cells may be prokaryotic cells such as E. coli,or eukaryotic cells such as yeast, insect, amphibian, or mammalian cellssuch as CHO, HeLa and the like, or plant cells. Both primary cells andcultured cell lines are included in this definition.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength, pH, and nucleic acid concentration) at which 50%of the probes complementary to the target sequence hybridize to thetarget sequence at equilibrium. Typically, stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.05 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of DNA duplex destabilizingagents such as formamide.

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Preferably, thepercent identity exists over a region of the sequence that is at leastabout 25 amino acids in length, more preferably over a region that is 50or 100 amino acids in length. This definition also refers to thecomplement of a test sequence, provided that the test sequence has adesignated or substantial identity to a reference sequence. Preferably,the percent identity exists over a region of the sequence that is atleast about 25 nucleotides in length, more preferably over a region thatis 50 or 100 nucleotides in length.

When percentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g,. charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Alternatively, when one includes such conservativesubstitutions in the comparison, a percent “similarity” can be noted, asopposed to a percent “identity”. Means for making this adjustment arewell known to those of skill in the art. The scoring of conservativesubstitutions can be calculated according to, e.g., the algorithm ofMeyers & Millers, Computer Applic. Biol. Sci. 4:11-17 (1988), e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

“Isogenic” refers to strains that have identical genomes but differ in asingle gene, be it resident on the chromosome or a plasmid.

The terms “isolated”, “purified”, or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. In anisolated gene, the nucleic acid of interest is separated from openreading frames which flank the gene of interest and encode proteinsother than the protein of interest. The term “purified” denotes that anucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, more preferably at least 95% pure, andmost preferably at least 99% pure.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include fluorescent proteins such as green, yellow, red or bluefluorescent proteins, radioisotopes such as ³²p, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available (e.g., the polypeptide of SEQ IDNO:2 can be made detectable, e.g., by incorporating a radio-label intothe peptide, and used to detect antibodies specifically reactive withthe peptide).

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker, or through ionic, van der Waals, orhydrogen bonds to a label such that the presence of the probe may bedetected by detecting the presence of the label bound to the probe.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and %SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

“Modulators,” “inhibitors,” and “activators of a target protein” referto modulatory molecules identified using in vitro and in vivo assay fortarget protein activity. Such assays include binding activity such asactin binding activity or binding to PIP₂. Samples or assays that aretreated with a candidate agent at a test and control concentration. Thecontrol concentration can be zero. If there is a change in targetprotein activity between the two concentrations, this change indicatesthe identification of a modulator. A change in activity, which can be anincrease or decrease, is preferably a change of at least 20% to 50%,more preferably by at least 50% to 75%, more preferably at least 75% to100%, and more preferably 150% to 200%, and most preferably is a changeof at least 2 to 10 fold compared to a control. Additionally, a changecan be indicated by a change in binding specificity or substrate.

“Multi-copy plasmid” refers to a plasmid having 10 to 30 copies presentin a cell.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences as well asthe sequence explicitly indicated. For example, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260)2605-2608(1985); Cassol et al. 1992; Rossolini et al. Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

“Nucleic acid probe or oligonucleotide” is defined as a nucleic acidcapable of binding to a target nucleic acid of complementary sequencethrough one or more types of chemical bonds, usually throughcomplementary base pairing, usually through hydrogen bond formation. Asused herein, a probe may include natural (i.e., A, G, C, or T) ormodified bases. In addition, the bases in a probe may be joined by alinkage other than a phosphodiester bond, so long as it does notinterfere with hybridization. Thus, for example, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledwith isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. Amino acids may be referred to herein by either their commonlyknown three letter symbols or by Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes, i.e., the one-letter symbols recommended by the IUPAC-IUB.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA box element. A promoter also optionally includes distal enhancer orrepressor elements which can be located as much as several thousand basepairs from the start site of transcription. A “constitutive” promoter isa promoter that is active under most environmental and developmentalconditions. An “inducible” promoter is promoter that is underenvironmental or developmental regulation. The term “operably linked”refers to a functional linkage between a nucleic acid expression controlsequence (such as a promoter, or array of transcription factor bindingsites) and a second nucleic acid sequence, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, antibodies raised toA. fumigatus profilin with the amino acid sequence encoded in SEQ IDNO:2 can be selected to obtain only those antibodies that arespecifically immunoreactive with A. fumigatus profilin and not withother proteins, except for polymorphic variants, orthologs, alleles, andclosely related homologues of A. fumigatus profilin. This selection maybe achieved by subtracting out antibodies that cross react withmolecules, for example, such as C. elegans unc-104 and human Kif1A byaffinity chromatography. A variety of immunoassay formats may be used toselect antibodies specifically immunoreactive with a particular protein.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

“Test composition” (used interchangeably herein with “candidate agent”and “test compound” and “test agent”) refers to a molecule orcomposition whose effect on the interaction between one or morecytoskeletal components it is desired to assay. The “test composition”can be any molecule or mixture of molecules, optionally in a carrier.

A “therapeutic” as used herein refers to a compound which is believed tobe capable of modulating the target protein in vivo which can haveapplication in both human and animal disease. Modulation of thecytoskeletal system would be desirable in a number of conditionsincluding, but not limited to antifungal agents and anti-inflammatoryagents.

“Variant” applies to both amino acid and nucleic acid sequences. Withrespect to particular nucleic acid sequences, conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG and GCT all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachdegenerate codon in a nucleic acid can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

Also included within the definition of target proteins of the presentinvention are amino acid sequence variants of wild-type target proteins.These variants fall into one or more of three classes: substitutional,insertional or deletional variants. These variants ordinarily areprepared by site specific mutagenesis of nucleotides in the DNA encodingthe target protein, using cassette or PCR mutagenesis or othertechniques well known in the art, to produce DNA encoding the variant,and thereafter expressing the DNA in recombinant cell culture. Varianttarget protein fragments having up to about 100-150 amino acid residuesmay be prepared by in vitro synthesis using established techniques.Amino acid sequence variants are characterized by the predeterminednature of the variation, a feature that sets them apart from naturallyoccurring allelic or interspecies variation of the target protein aminoacid sequence. The variants typically exhibit the same qualitativebiological activity as the naturally occurring analogue, althoughvariants can also be selected which have modified characteristics.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to about 20 amino acids,although considerably longer insertions may be tolerated. Deletionsrange from about 1 to about 20 residues, although in some cases,deletions may be much longer.

Substitutions, deletions, and insertions or any combinations thereof maybe used to arrive at a final derivative. Generally, these changes aredone on a few amino acids to minimize the alteration of the molecule.However, larger characteristics may be tolerated in certaincircumstances.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984)).

II. The Target Protein

The present invention provides for the first time a nucleic acidencoding A. fumigatus profilin. This protein is a member of the profilinsuperfamily. More specifically, the A. fumigatus profilin sequence ofFIG. 2 shares approximately 30-40% identity to S. pombe profilin;Candida albicans profilin; and S. cerevisiae profilin over 126 aminoacids.

In one aspect, A. fumigatus profilin can be defined by having at leastone or preferably more than one of the following functional andstructural characteristics. Functionally, A. fumigatus profilin will becapable of binding actin and PIP₂.

The novel nucleotides sequences provided herein encode A. fumigatusprofilin or fragments thereof. Thus, in one aspect, the nucleic acidsprovided herein are defined by the novel proteins provided herein. Theprotein provided herein comprises an amino acid sequence which has oneor more of the following characteristics: greater than 70% sequenceidentity with SEQ ID NO:2, preferably greater than 80%, more preferablygreater than 90%, more preferably greater than 95% or, in anotherembodiment, has 98 to 100% sequence identity with SEQ ID NO:2. Asdescribed above, when describing the nucleotide in terms of SEQ ID NO:1,the sequence identity may be slightly lower due to the degeneracy in thegenetic code. Also included within the definition of the target proteinsare amino acid sequence variants of wild-type target proteins.

Portions of the A. fumigatus profilin nucleotide sequence may be used toidentify polymorphic variants, orthologs, alleles, and homologues of A.fumigatus profilin. This identification can be made in vitro, e.g.,under stringent hybridization conditions and sequencing, or by using thesequence information in a computer system for comparison with othernucleotide sequences. Sequence comparison can be performed using any ofthe sequence comparison algorithms discussed below, with PILEUP as apreferred algorithm.

As will be appreciated by those in the art, the target proteins can bemade in a variety of ways, including both synthesis de novo and byexpressing a nucleic acid encoding the protein.

Target proteins of the present invention may also be modified in a wayto form chimeric molecules comprising a fusion of a target protein witha tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino or carboxyl terminus of the target protein. Provision of theepitope tag enables the target protein to be readily detected, as wellas readily purified by affinity purification. Various tag epitopes arewell known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the influenza HA tagpolypeptide and its antibody 12CA5 (see, Field et al. (1988) Mol. Cell.Biol. 8:2159); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (see, Evans et al., (1985) Molecular and CellularBiology, 5:3610); and the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (see, Paborsky et al., (1990) Protein Engineering,3:547). Other tag polypeptides include the Flag-peptide (see, Hopp etal. (1988) BioTechnology 6:1204); the KT3 epitope peptide (see, Martineet al. (1992) Science, 255:192); tubulin epitope peptide (see, Skinner(1991) J. Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag(see, Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393.

The biological activity of any of the peptides provided herein can beroutinely confirmed by the assays known in the art. In one embodiment,polymorphic variants, alleles, and orthologs, homologues of A. fumigatusprofilin are confirmed by using actin or PIP₂ assays as known in theart.

The isolation of biologically active A. fumigatus profilin for the firsttime provides a means for assaying for modulators of this protein.Biologically active A. fumigatus profilin is useful for identifyingmodulators of A. fumigatus profilin or fragments thereof. In vivo assaysand uses are provided herein as well. Also provided herein are methodsof identifying candidate agents which bind to A. fumigatus profilin andportions thereof.

As further described herein, a wide variety of assays, therapeutic anddiagnostic methods are provided herein which utilize the novel compoundsdescribed herein. The uses and methods provided herein, as furtherdescribed below have in vivo, in situ, and in vitro applications, andcan be used in medicinal, veterinary, agricultural and research basedapplications.

III. Isolation of the Gene Encoding A. fumigatus Profilin

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from mass spectroscopy, from sequenced proteins, fromderived amino acid sequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter etal., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonculeotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.225:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences Encoding A.fumigatus Profilin

In general, the nucleic acid sequences encoding A. fumigatus profilinand related nucleic acid sequence homologs are cloned from cDNA andgenomic DNA libraries or isolated using amplification techniques witholigonucleotide primers. Alternatively, expression libraries can be usedto clone A. fumigatus profilin and A. fumigatus profilin homologues bydetected expressed homologues immunologically with antisera or purifiedantibodies made against A. fumigatus profilin that also recognize andselectively bind to the A. fumigatus profilin homologue. Finally,amplification techniques using primers can be used to amplify andisolate A. fumigatus profilin from DNA or RNA. Amplification techniquesusing degenerate primers can also be used to amplify and isolate A.fumigatus profilin homologues. Amplification techniques using primerscan also be used to isolate a nucleic acid encoding A. fumigatusprofilin. These primers can be used, e.g., to amplify a probe of severalhundred nucleotides, which is then used to screen a library forfull-length A. fumigatus profilin.

Appropriate primers and probes for identifying the gene encodinghomologues of A. fumigatus profilin in other species are generated fromcomparisons of the sequences provided herein. As described above,antibodies can be used to identify A. fumigatus profilin homologues. Forexample, antibodies made to the conserved actin-binding domain of A.fumigatus profilin or to the whole protein are useful for identifying A.fumigatus profilin homlogues.

To make a cDNA library, one should choose a source that is rich in themRNA of choice, e.g., A. fumigatus profilin. The genomic library isusually contained in, for example, a modified bacteriophage lambda or E.coli plasmid (Sambrook et al. supra). The cDNA library may be containedin such vectors as λgt10, λgt11, or lambda ZAP. The mRNA is convertedinto cDNA using reserve transcriptase, made double stranded, ligatedinto a recombinant vector, and introduced into a recombinant host forpropagation, screening and cloning. Methods for making and screeningcDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene 25:263-269); Sambrook et al., supra; Ausubel et al., supra).

An alternative method of isolating A. fumigatus profilin nucleic acidand its homologues combines the use of synthetic oligonucleotide primersand amplification of an RNA or DNA template (see U.S. Pat. Nos.4,683,195 and 4,683,202; PCR Protocols: A guide to Methods andApplications (Innis et al., eds. 1990)). Methods such as polymerasechain reaction and ligase chain reaction can be used to amplify nucleicacid sequences of A. fumigatus profilin directly from mRNA, from cDNA,from genomic libraries or cDNA libraries. Degenerate oligonucleotidescan be designed to amplify A. fumigatus profilin homologues using thesequences provided herein. Restriction endonuclease sites can beincorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of A.fumigatus profilin encoding mRNA in physiological samples, for nucleicsequencing or for other purposes. Genes amplified by the PCR reactioncan be purified from agarose gels and cloned into an appropriate vector.

Gene expression of A. fumigatus profilin can also be analyzed bytechniques known in the art, e.g., reverse transcription andamplification of mRNA, isolation of total RNA or poly A+RNA, northernblotting, dot blotting, in situ hybridization, RNase protection,quantitative PCR, and the like.

Synthetic oligonucleotides can be used to construct recombinant A.fumigatus profilin genes for use as probes or for expression of protein.This method is performed using a series of overlapping oligonucleotidesusually 40-120 bp in length, representing both the sense and nonsensestrands of the gene. These DNA fragments are then annealed, ligated andcloned. Alternatively, amplification techniques can be used with preciseprimers to amplify a specific subsequence of the A. fumigatus profilingene. The specific subsequence is then ligated into an expressionvector.

The gene for A. fumigatus profilin is typically cloned into intermediatevectors before transformation into prokaryotic or eukaryotic cells forreplication and/or expression. The intermediate vectors are typicallyprokaryote vectors or shuttle vectors.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding A. fumigatus profilin, it is important to construct anexpression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the A. fumigatus profilinprotein are available in, e.g., E. coli, Bacillus sp., and Salmonella(Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature302:543-545 (1983). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems are well known in the art andare also commercially available.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the A. fumigatusprofilin encoding nucleic acid in host cells. A typical expressioncassette thus contains a promoter operably linked to the nucleic acidsequence encoding A. fumigatus profilin and signals required forefficient polyadenylation of the transcript, ribosome binding sites, andtranslation termination. The nucleic acid sequence encoding A. fumigatusprofilin may typically be linked to a cleavable signal peptide sequenceto promote secretion of the encoded protein by the transformed cell.Such signal peptides would include, among others, the signal peptidesfrom tissue plasminogen activator, insulin, and neuron growth factor,and juvenile hormone esterase of Heliothis virescens. Additionalelements of the cassette may include enhancers and, if genomic DNA isused as the structural gene, introns with functional splice donor andacceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc or histidine tags.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, cytomegalovirus vectors, papilloma virus vectors, and vectorsderived from Epstein Bar virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe SV40 early promoter, SV40 late promoter, CMV promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a A. fumigatus profilinencoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection or transformation methods are used to producebacterial, mammalian, yeast or insect cell lines that express largequantities of A. fumigatus profilin protein, which are then purifiedusing standard techniques (see, e.g., Colley et al., J. Biol. Chem.264:17619-17622 (1989); Guide to Protein Purification, in Methods inEnzymology, vol.182 (Deutscher ed., 1990)).

Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.,132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology,101:347-362 (Wu et al., eds, 1983). Any of the well known procedures forintroducing foreign nucleotide sequences into host cells may be used.These include the use of calcium phosphate transfection, polybrene,protoplast fusion, electroporation, liposomes, microinjection, plasmavectors, viral vectors and any of the other well known methods forintroducing cloned genomic DNA, cDNA, synthetic DNA or other foreigngenetic material into a host cell (see, e.g., Sambrook et al., supra).It is only necessary that the particular genetic engineering procedureused be capable of successfully introducing at least one gene into thehost cell capable of expressing A. fumigatus profilin.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring-expression ofA. fumigatus profilin, which is recovered from the culture usingstandard techniques identified below.

IV. Purification of A. fumigatus Profilin Protein

Either naturally occurring or recombinant A. fumigatus profilin can bepurified for use in functional assays. In a preferred embodiment, thetarget proteins are purified for use in the assays to providesubstantially pure samples.

The target proteins may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological, and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, chromatofocussing, selectiveprecipitation with such substances as ammonium sulfate;and others (see,e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S.Pat. No. 4,673,641; Ausubel et al. supra; and Sambrook et al., supra).For example, the target protein can be purified using a standardanti-target antibody column. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.A preferred method of purification is use of Ni-NTA agarose (Qiagen) incombination with polyhistine-tagged proteins.

The expressed protein can be purified by standard chromatographicprocedures to yield a purified, biochemically active protein. Theactivity of any of the peptides provided herein can be routinelyconfirmed by the assays known in the art.

A. Purification of A. fumigatus Profilin From Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a preferred method ofexpression. Bacteria are grown according to standard procedures in theart. Fresh or frozen bacteria cells are used for isolation of protein.Alternatively, it is possible to purify A. fumigatus profilin frombacteria periplasm. After A. fumigatus profilin is exported into theperiplasm of the bacteria, the periplasmic fraction of the bacteria canbe isolated by cold osmotic shock in addition to other methods known toskill in the art. To isolate recombinant proteins from the periplasm,the bacterial cells are centrifuged to form a pellet. The pellet isresuspended in a buffer containing 20% sucrose. To lyse the cells, thebacteria are centrifuged and the pellet is resuspended in ice-cold 5 mMMgSO₄ and kept in an ice bath for approximately 10 minutes. The cellsuspension is centrifuged and the supernatant decanted and saved. Therecombinant proteins present in the supernatant can be separated fromthe host proteins by standard separation techniques well known to thoseof skill in the art. Suitable purification schemes for some specifickinesins are outlined in U.S. Ser. No. 09/295,612, filed Apr. 20, 1999,hereby expressly incorporated herein in its entirety for all purposes.

B. Standard Protein Separation Techniques For Purifying A. fumigatusProfilin

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of A. fumigatus profilin can be used to isolated itfrom proteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

A. fumigatus profilin can also be separated from other proteins on thebasis of its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

V. Immunological Detection of A. fumigatus Profilin

In addition to the detection of A. fumigatus profilin genes and geneexpression using nucleic acid hybridization technology, one can also useimmunoassays to detect A. fumigatus profilin. Immunoassays can be usedto qualitatively or quantitatively analyze A. fumigatus profilin. Ageneral overview of the applicable technology can be found in Harlow &Lane, Antibodies: A Laboratory Manual (1988).

A. Antibodies to A. fumigatus Profilin

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with A. fumigatus profilin are known to those of skill inthe art (see, e.g., Coligan, Current Protocols in Immunology (1991);Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles andPractice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497(1975). Such techniques include antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors, as well as preparation of polyclonal and monoclonal antibodiesby immunizing rabbits or mice (see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)).

A number of A. fumigatus profilin comprising immunogens may be used toproduce antibodies specifically reactive with A. fumigatus profilin. Forexample, recombinant A. fumigatus profilin or a antigenic fragmentthereof, is isolated as described herein. Recombinant protein can beexpressed in eukaryotic or prokaryotic cells as described above, andpurified as generally described above. Recombinant protein is thepreferred immunogen for the production of monoclonal or polyclonalantibodies. Alternatively, a synthetic peptide derived from thesequences disclosed herein and conjugated to a carrier protein can beused an immunogen. Naturally occurring protein may also be used eitherin pure or impure form. The product is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies may be generated, for subsequent use in immunoassays tomeasure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to A. fumigatus profilin.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-A. fumigatusprofilin proteins or even other homologous proteins from other organisms(e.g., fungal or vertebrate profilins), using a competitive bindingimmunoassay. Specific polyclonal antisera and monoclonal antibodies willusually bind with a K_(d) of at least about 0.1 mM, more usually atleast about 1 μM, preferably at least about 0.1 μM or better, and mostpreferably, 0.01 μM or better.

Once A. fumigatus profilin specific antibodies are available, A.fumigatus profilin can be detected by a variety of immunoassay methods.For a review of immunological and immunoassay procedures, see Basic andClinical Immunology (Stites & Terr eds., 7th ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio ed., 1980); and Harlow & Lane, supra.

B. Binding Assays

Antibodies can be used for treatment or to identify the presence of A.fumigatus profilin having the sequence identity characteristics asdescribed herein. Additionally, antibodies can be used to identifymodulators of the interaction between the antibody and A. fumigatusprofilin as further described below. While the following discussion isdirected toward the use of antibodies in the use of binding assays, itis understood that the same general assay formats such as thosedescribed for “non-competitive” or “competitive” assays can be used withany compound which binds to A. fumigatus profilin such as microtubulesor the compounds described in Ser. No. 60/070,772.

In a preferred embodiment, A. fumigatus profilin is detected and/orquantified using any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Methods in Cell Biology Volume 37: Antibodies in Cell Biology(Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds.,7th ed. 1991). Immunological binding assays (or immunoassays) typicallyuse an antibody that specifically binds to a protein or antigen ofchoice (in this case the A. fumigatus profilin or antigenic subsequencethereof). The antibody (e.g., anti-A. fumigatus profilin) may beproduced by any of a number of means well known to those of skill in theart and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled A. fumigatus profilinpolypeptide or a labeled anti-A. fumigatus profilin antibody.Alternatively, the labeling agent may be a third moiety, such asecondary antibody, that specifically binds to the antibody/A. fumigatusprofilin complex (a secondary antibody is typically specific toantibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see generallyKronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 4° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting A. fumigatus profilin in samples may beeither competitive or noncompetitive. Noncompetitive immunoassays areassays in which the amount of antigen is directly measured. In onepreferred “sandwich” assay, for example, the anti-A. fumigatus profilinantibodies can be bound directly to a said substrate on which they areimmobilized. These immobilized antibodies then capture A. fumigatusprofilin present in the test sample. A. fumigatus profilin is thusimmobilized is then bound by a labeling agent, such as a second A.fumigatus profilin antibody bearing a label. Alternatively, the secondantibody may lack a label, but it may, in turn, be bound by a labeledthird antibody specific to antibodies of the species from which thesecond antibody is derived. The second or third antibody is typicallymodified with a detectable moiety, such as biotin, to which anothermolecule specifically binds, e.g., streptavidin, to provide a detectablemoiety.

Competitive Assay Formats

In competitive assays, the amount of A. fumigatus profilin present inthe sample is measured indirectly by measuring the amount of a known,added (exogenous) A. fumigatus profilin displaced (competed away) froman anti-A. fumigatus profilin antibody by the unknown A. fumigatusprofilin present in a sample. In one competitive assay, a known amountof A. fumigatus profilin is added to a sample and the sample is thencontacted with an antibody that specifically binds to A. fumigatusprofilin. The amount of exogenous A. fumigatus profilin bound to theantibody is inversely proportional to the concentration of A. fumigatusprofilin present in the sample. In a particularly preferred embodiment,the antibody is immobilized on a solid substrate. The amount of A.fumigatus profilin bound to the antibody may be determined either bymeasuring the amount of A. fumigatus profilin present in a A. fumigatusprofilin/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of A. fumigatus profilin maybe detected by providing a labeled A. fumigatus profilin molecule.

Cross-reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NO:2 can be immobilized to a solid support.Proteins (e.g., other fungal or vertebrate profilins) are added to theassay that compete for binding of the antisera to the immobilizedantigen. The ability of the added proteins to compete for binding of theantisera to the immobilized protein is compared to the ability of A.fumigatus profilin encoded by SEQ ID NO:2 to compete with itself. Thepercent crossreactivity for the above proteins is calculated, usingstandard calculations. Those antisera with less than 10% crossreactivitywith each of the added proteins listed above are selected and pooled.The cross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologues.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps the protein of this invention, to the immunogenprotein (i.e., A. fumigatus profilin of SEQ ID NO:2). In order to makethis comparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required to inhibit 50% of binding isless than 10 times the amount of the protein encoded by SEQ ID NO:2 thatis required to inhibit 50% of binding, then the second protein is saidto specifically bind to the polyclonal antibodies generated to a A.fumigatus profilin immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of A. fumigatus profilin in the sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind A. fumigatus profilin. The anti-A. fumigatusprofilin antibodies specifically bind to the A. fumigatus profilin onthe solid support. These antibodies may be directly labeled oralternatively may be subsequently detected using labeled antibodies(e.g., labeled sheep anti-mouse antibodies) that specifically bind tothe anti-A. fumigatus profilin antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), colorimetric labels such as colloidalgold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.) or other labels that can be detected by massspectroscopy, NMR spectroscopy, or other analytical means known in theart.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize A. fumigatusprofilin, or secondary antibodies that recognize anti-A. fumigatusprofilin.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of the Target Protein

The present invention provides methods to identify candidate agents thatbind to a target protein or act as a modulator of the bindingcharacteristics or biological activity of a target protein. In oneembodiment, the method is performed in plurality simultaneously. Forexample, the method can be performed at the same time on multiple assaymixtures in a multi-well screening plate. Thus, in one aspect, theinvention provides a high throughput screening system.

More specifically, the methods of the present invention can be used toidentify inhibitors of profilin that have antifungal activity and thattherefore provide the starting point for maturing a candidate compoundfor clinical development. These methods can be used to identifyinhibitors that show biochemical inhibition of bacterially expressedAspergillus fumigatus and Candida albicans profilin in vitro andcounterscreened against human profilin. In a particularly preferredembodiment, the method also comprises the further step of measuring thetoxicity of the compounds of interest on mammalian cells.

Genes encoding A. fumigatus profilin were cloned by amplifying fragmentsof Aspergillus nidulans ESTs, then screening an Aspergillus fumigatuscDNA library at low stringency. The protein was expressed in E. coli,and was purified to homogeneity. Using this method, tens to hundreds ofmilligrams of purified protein per liter of culture, can be obtained.The protein exhibited the biochemical activities previously demonstratedfor homologs isolated from other organisms. Candida albicans proteinswere obtained by cloning the respective genes using publically availablesequence information, designing appropriate primers, performing PCR, andexpressing these cloned genes as described above. Candida albicansprofilin was purified to homogeneity as described for A. fumigatusprofilin. See, e.g., PCT Publication WO 97/31104, which is incorporatedherein by reference for all purposes.

Assays for the interaction of A. fumigatus and any other profilin withactin in 384-well plates using a fluorescence as the detection meanshave been developed. The assays employ pyrene-labeled actin (Pollard(1984) J Cell Biol 99:769-777). When pyrene actin assembles, the quantumyield of fluorescence increases 25-fold (FIG. 3). This very robustsignal can be exploited to an assay for modulators of profilin. Chickenskeletal actin was used because it can be purified in quantities neededfor high throughput screening; it is 90% identical in amino acidsequence to fungal actin, and it can assemble into copolymers withfungal actin. Pyrene-labeled skeletal actin has been prepared andcharacterized as to its polymerization properties as well as for itsinteraction with fungal profilin.

The kinetics of actin assembly in the absence and presence of profilincan be monitored. Actin assembly proceeds in three phases. There is alag phase during which time seeds form from three actin monomers. Thisis followed by an elongation phase during which filaments are extendedfrom seeds. Finally, at steady state, the polymer concentration isconstant, although filaments treadmill. Although actin bound to profilincan elongate the barbed ends of pre-existing actin filaments, profilinvery potently inhibits the nucleation of filaments de novo (Pollard etal. (1984) Biochem. 23:6631-6641.). FIG. 3 shows inhibition ofpyrene-actin nucleation by A. fumigatus profilin in a 384-well plateassay. An inhibitor of the interaction of profilin with actin shouldovercome this inhibition of actin nucleation and cause a pronouncedincrease in pyrene fluorescence, ideally to levels approaching that seenin the absence of profilin.

Accordingly, the present invention provides for a high throughput methodfor the identification of small molecules that inhibit or modulateprofilin interactions with actin. Several biochemical assays (many ofwhich assess key in vivo functions of the proteins) exist for profilin(Kreis, T and Vale, R (eds) (1999) Guidebook to the Cytoskeletal andMotor Proteins. Oxford University Press, Oxford). For example,etheno-ATP fluorescence can be used to screen for compounds that inhibitprofilin's enhancement of actin nucleotide exchange; genetic evidencesuggests that this activity is critical in vivo (Wolven et al., 2000).

C. Candidate Agents

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. In a preferred embodiment,the candidate agents are organic chemical moieties, a wide variety ofwhich are available in the literature.

VII. Applications

Fungal infections which can be inhibited or treated with compositionsprovided herein include but are not limited to: Candidiasis includingbut not limited to onchomycosis, chronic mucocutaneous candidiasis, oralcandidiasis, epiglottistis, esophagitis, gastrointestinal infections,genitourinary infections, for example, caused by any Candida species,including but not limited to Candida albicans, Candida tropicalis,Candida (Torulopsis) glabrata, Candida parapsilosis, Candida lusitaneae,Candida rugosa and Candida pseudotropicalis; Aspergillosis including butnot limited to granulocytopenia caused for example, by, Aspergillus spp.including but not limited to A. fumigatus, Aspergillus flavus,Aspergillus niger and Aspergillus terreus; Zygomycosis, including butnot limited to pulmonary, sinus and rhinocerebral infections caused by,for example, zygomycetes such as Mucor. Rhizopus spp., Absidia,Rhizomucor, Cunningamella, Saksenaea, Basidobolus and Conidobolus;Cryptococcosis, including but not limited to infections of the centralnervous system—meningitis and infections of the respiratory tract causedby, for example, Cryptococcus neoformans; Trichosporonosis caused by,for example, Trichosporon beigelii; Pseudallescheriasis caused by, forexample, Pseudallescheria boydii; Fusarium infection caused by, forexample, Fusarium such as Fusarium solani, Fusarium moniliforme andFusarium proliferatum; and other infections such as those caused by, forexample, Penicillium spp. (generalized subcutaneous abscesses),Drechslera, Bipolaris, Exserohilum spp., Paecilomyces lilacinum,Exophila jeanselmei (cutaneous nodules), Malassezia furfur(folliculitis), Alternaria (cutaneous nodular lesions), Aureobasidiumpullulans (splenic and disseminated infection), Rhodotorula spp.(disseminated infection), Chaetomium spp. (empyema), Torulopsis candida(fungemia), Curvularia spp. (nasopharnygeal infection), Cunninghamellaspp. (pneumonia), H. Capsulatum, B. dermatitidis, Coccidioides immitis,Sporothrix schenckii and Paracoccidioides brasiliensis, Geotrichumcandidum (disseminated infection).

Treating “fungal infections” as used herein refers to the treatment ofconditions resulting from fungal infections. Therefore, one may treat,for example, pneumonia, nasopharnygeal infections, disseminatedinfections and other conditions listed above and known in the art byusing the compositions provided herein. In preferred embodiments,treatments and sanitization of areas with the compositions providedherein are provided to immunocompromised patients or areas where thereare such patients. Wherein it is desired to identify the particularfungi resulting in the infection, techniques known in the art may beused, see, for example, Musial et al., Clin. Microbiol. Rev., AmericanSociety for Microbiology, 349-64 (1998), incorporated herein byreference.

Accordingly, the compositions of the invention are administered tocells. By “administered” herein is meant administration of atherapeutically effective dose of the candidate agents of the inventionto a cell either in cell culture or in a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. By “cells” herein is meantalmost any cell in which mitosis or meiosis or in which cell morphology(e.g., filamentous hyphal growth, etc.) can be altered.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and other organisms.Thus the methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

Candidate agents having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %. The agents maybe administered alone or incombination with other treatments, i.e., radiation, or otherchemotherapeutic agents.

In a preferred embodiment, the pharmaceutical compositions are in awater soluble form, such as pharmaceutically acceptable salts, which ismeant to include both acid and base addition salts.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The administration of the candidate agents of the present invention canbe done in a variety of ways as discussed above, including, but notlimited to, orally, subcutaneously, intravenously, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,intrathecally, vaginally, rectally, or intraocularly. In some instances,for example, in the treatment of wounds and inflammation, the candidateagents may be directly applied as a solution or spray.

One of skill in the art will readily appreciate that the methodsdescribed herein also can be used for diagnostic applications. Adiagnostic as used herein is a compound or method that assists in theidentification and characterization of a health or disease state inhumans or other animals. More specifically, antibodies whichspecifically bind A. fumigatus profilin may be used for the diagnosis ofdisorders characterized by expression of A. fumigatus profilin or inassays to monitor patients being treated with A. fumigatus profilin, oragonists, antagonists, or inhibitors of A. fumigatus profilin.Diagnostic assays include methods which utilize the antibody and a labelto detect A. fumigatus profilin in human body fluids or in extracts ofcells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecules. A wide variety of reporter molecules are knownin the art and may be used.

A variety of protocols for measuring A. fumigatus profilin, includingELISAs, RIAs, and FACS, are known in the art and provide a basis fordiagnosing altered or abnormal levels of A. fumigatus profilinexpression. Normal or standard values for A. fumigatus profilinexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toA. fumigatus profilin under conditions suitable for complex formation.The amount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of A. fumigatusprofilin expressed in subject, control, and disease samples frombiopsied tissues can be compared with the standard values. Deviationbetween standard and subject values establishes the parameters fordiagnosing disease.

In another embodiment of the invention, the polynucleotides encoding A.fumigatus profilin may be used for diagnostic purposes. Thepolynucleotides which may be used include oligonucleotide sequences,complementary RNA and DNA molecules, and PNAs. The polynucleotides maybe used to detect and quantitate gene expression in biopsied tissues inwhich A. fumigatus profilin may be correlated with disease. Thediagnostic assay may be used to determine absence, presence, and excessexpression of A. fumigatus profilin, and to monitor regulation of A.fumigatus profilin levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences encodingA. fumigatus profilin or closely related molecules may be used. Thespecificity of the probe, whether it's made from a highly specificregion or from a less specific region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding A. fumigatus profilin, allelic variants, or relatedsequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of the A.fumigatus profilin encoding sequences. The hybridization probes of thesubject invention may be DNA or RNA and may be derived from the sequenceof SEQ ID NO:1 or from genomic sequences including promoters, enhancers,and introns of the A. fumigatus profilin gene.

Means for producing specific hybridization probes for DNAs encoding A.fumigatus profilin include the cloning of polynucleotide sequencesencoding A. fumigatus profilin or derivatives thereof into vectors forthe production of mRNA probes. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by means of the addition of the appropriate RNA polymerases andthe appropriate labeled nucleotides. Hybridization probes may be labeledby a variety of reporter groups, for example, by radionuclides such as³²P or ³⁵S, or by enzymatic labels, such as alkaline phosphatase coupledto the probe via avidin/biotin coupling systems and the like.

In a particular aspect, the nucleotide sequences encoding A. fumigatusprofilin may be useful in assays that detect the presence of associateddisorders. The nucleotide sequences encoding A. fumigatus profilin maybe labeled by standard methods and added to a fluid or tissue samplefrom a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal is significantly altered in comparison toa control sample then the presence of altered levels of nucleotidesequences encoding A. fumigatus profilin in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding A. fumigatus profilin may involve the use of PCR.These oligomers may be chemically synthesized, generated enzymatically,or produced in vitro based on SEQ ID NO:1. Oligomers will preferablycontain a fragment of a polynucleotide encoding A. fumigatus profilin,or a fragment of a polynucleotide complementary to the polynucleotideencoding A. fumigatus profilin, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences. Quantitative PCRas practiced by those of skill in the art can be used to detect relativelevels of A. fumigatus mRNA in a test sample.

Methods which may be used to quantitate the expression of A. fumigatusprofilin include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and interpolating resultsfrom standard curves. The speed of quantitation of multiple samples maybe accelerated by running the assay in an ELISA format where theoligomer of interest is presented in various dilutions and aspectrophotometer or calorimetric response gives rapid quantitation.

One of skill in the art will readily appreciate that the methodsdescribed herein also can be used for diagnostic applications. Adiagnostic as used herein is a compound or method that assists in theidentification and characterization of a health or disease state inhumans or other animals.

The present invention also provides for kits for screening formodulators of the target protein. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: biologically active targetprotein, reaction tubes, and instructions for testing activity of thetarget protein. Preferably, the kit contains biologically active targetprotein. A wide variety of kits and components can be prepared accordingto the present invention, depending upon the intended user of the kitand the particular needs of the user. For example, the kit can betailored for actin binding assays.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

1. Preparation on Actin Acetone Powder From Chicken Muscle

Mince 2 kg of fresh, boneless, skinless chicken breast in a pre-chilledmeat grinder (coarse grind). Extract with moderate stirring in 4 L of0.1 M KCL, 0.15 M Potassium Phosphate, pH. 6.5 for 10 mins at 4° C.Centrifuge at 5,000 rpm for 10 mins at 4° C. Extract the pellets withstrirring in 4 L of 0.05 M NaHCO₃ for exactly 5 minutes at 4° C. Repeat.Extract the pellets once with stirring in 4 L of 1 mM EDTA, pH 7.0 at 4°C. Extract the pellets once with stirring in 4 L of nano-pure dH₂O for10 mins. at 4° C. Carefully collect the pellets (part solid andgelatinous) into an 8 L beaker. Extract 5 times with stirring in 4 L ofpre-chilled acetone for 10 mins. at room temperature in a fume hood. Foreach extraction, decant the top layer of acetone, then pass theremaining protein precipitate through one-layer of cheesecloth. Gentlysqueeze the residual acetone without adding excessive pressure. Layerthe protein powder (residue) unto a glass pyrex dish lined withcheesecloth. Cover with another layer of cheesecloth and dry in the fumehood overnite. Store the residue in a wide mouth plastic bottle at −20°C. overnite. From 4 kg of chicken breast meat, a total of 450 g of actinacetone powder was generated.

2. “Clean” Chicken Breast Actin

Extract chicken breast acetone powder (as prepared above) in Buffer A:Use 200 ml of cold buffer A per 10 g of powder. Collect the extract bypassing and squeezing the mixture through several layers ofpre-sterilized cheesecloth. Re-extract the residue in the same volume ofbuffer A. Combine the extracts. Discard the extracted powder. Spin inJLA10 rotor(s) at 10 K rpm for 1 hr at 4° C. Collect the supernatantthrough 2 layers of pre-sterilized cheesecloth. Add Na₂ATP to 0.2 mM andMgCL₂ to 50 mM. Stir on stir plate for 60 mins at 4° C. to allow actinto polymerize and form para-crystals. Note: Na₂ATP is at 100 mM stock(pH 8,0)and MgCL₂ at 1M (pH 8.0) Slowly add solid KCL to 0.6 M (45 g perL of buffer) and stir at 4° C. for 30 mins. Spin in JLA 10 rotor(s) at10K rpm for 1 hr at 4° C. Discard the supernatant and quickly rinse thesurface of the pellets with cold buffer A. Dischard the wash. Add asmall amount of cold buffer A (about 25-30 ml) to each bottle to softenthe pellets. Resuspend vigorously by shaking the capped bottles by hand.Combine the pellets into a clean bottle and adjust the volume withbuffer A so that 3ml of buffer A is used per gram of original powder.Homogenize by using a large douncer (pestle B) on ice. Dialyze againstbuffer A with 4 changes over a 48 hour period at 4° C. with stirring.Collect the dialyzed Actin and spin at 40K rpm using a 45Ti rotor at 4°C. for 1 hr. Collect the supernatant (G-Actin). To polymerize G-actinfor storage add KCL to 50 mM (from 3 M stock), MgCl₂ to 1 mM and NaN₃ to0.02% (from 10% stock).

Buffer A

2 mM Tris

0.2 mM CaCl₂

0.005% NaN₃

0. 5 mM B-mercaptoethanol (36 ul per liter)

0.2 mM Na₂ATP

pH 8.0 when making 1×solution

3. Pyrene-Actin

Start with “Clean” actin as prepared above. Adjust the concentration ofclean actin to 40-∵uM with G- buffer. Dialyze several times againstG-buffer with No DTT or 2-mercaptoethanol over night or longer. Afterdialysis, transfer actin into a foil-wrapped tube or bottle. In rapidsuccession, add the following into the bottle:

1. MgCl₂ to 2 mM (use 1 M stock)

2. *Pyrene to 45 uM with gentle mixing (use 60×stock; see below)

3. KCL to 0.1 M (use 3 M stock)

Incubate in the dark for 90 minutes at 20° C. with gentle shaking orstirring. Pellet the F-Actin (polymerized) in a TLA100.3 rotor. At 90 Krpm for 15 mins. Combine the pellets and resuspend in G-buffer to afinal concentration of about 60 uM. Dialyze against several liters ofG-buffer with DTT or 2-mercaptoethanol over night or for a few days at4° C. in the dark. Do a clearing spin to remove the precipitated pyreneand denatured actin at 90 K for 15 mins or 45 K for 1 hour. Tranfer theG-actin into a sterile bottle on ice and in the dark. Drop freeze inliquid nitrogen and store pellets in a dark (foiled) bottle at −80° C.

Measure pyrene concentration at 339 nM (E=28,100/cm/M), actinconcentration after pyrene labeling (OD_(290−0.33)×OD₃₃₉)/24.9

*Pyrene Iodoacetamide: Dissolve pyrene in anhydrous DMSO to 1 mg/ml (2.6mM). This becomes a 60×dilution which you will use to get a finalconcentration of 45 uM in step. No. 5. (Molecular probes P-29, N-1(pyrene) iodoacetamide)

Optimized profilin Assays: Inhibition of Actin Assembly

Assay Conditions (for Plate Reader Experiments, 50 ul per well)

Thaw Actin, Pyrene-Actin and Profilin pellets on ice or at room temp.(approx. 500 ul per sample), then on ice at all times. Ultra-centrifuge(Beckman) Actin and Pyrene-actin at 100,000 rpm for 30 min to 1 hour at4° C. Remove 75% of the top layer and transfer to a sterile tube. Takereadings at OD₂₉₀. Calculate concentrations in uM using OD₂₉₀ of 0.6=25uM (or 1 mg/ml). Actin concentrations are kept constant at 6 uM andPyrene-actin at 2 uM. 25 ul of salts will be added last. Vary profilinconcentrations from 0 uM to 5.0 or 7.5 uM.

Add 25 ul of sample into each well. Add 25 ul of 2X initiation salts toeach well. Take Pyrene-Kinetic readings (Fluorostar setting) at 150cycles, 360 to 407 nM.

G Buffer

5 mM Tris-Cl, pH 8.0

0.2 mM ATP

0.1 mM CaCl2

0.5 mM DTT

10×Initiation Salts

500 mM KCL

10 mM MgCl2

100 mM Tris-Cl, pH 8.0

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety.

2 1 396 DNA Aspergillus fumigatus 1 atgggtcagc actcggcagt ttggcaaggatatgttgatt ccagtttgat gggctccggc 60 cagtttgaca aggccgccat tctgagctacgacttctccg acgtcgaggc ccagtcccct 120 actttccaga tctcgaagga ggaaattgccgggctgaagg ctgctttcga caagcctggc 180 agtgctttcg aaaccggttt cgtggtcggaggagacaagt tcgtcgccat caaggctgat 240 gatcgcagtc tctacggcaa gaagggcaaagagggtatcg tcgtcgtcaa ggccgtctct 300 tgcgtcatgg tggcccatca tggcgaggccgttcagacca ccaacgctgc aaccgttgtc 360 gagaacctcg tcgactacat caacaacccccggtag 396 2 131 PRT Aspergillus fumigatus 2 Met Gly Gln His Ser Ala ValTrp Gln Gly Tyr Val Asp Ser Ser Leu 1 5 10 15 Met Gly Ser Gly Gln PheAsp Lys Ala Ala Ile Leu Ser Tyr Asp Phe 20 25 30 Ser Asp Val Glu Ala GlnSer Pro Thr Phe Gln Ile Ser Lys Glu Glu 35 40 45 Ile Ala Gly Leu Lys AlaAla Phe Asp Lys Pro Gly Ser Ala Phe Glu 50 55 60 Thr Gly Phe Val Val GlyGly Asp Lys Phe Val Ala Ile Lys Ala Asp 65 70 75 80 Asp Arg Ser Leu TyrGly Lys Lys Gly Lys Glu Gly Ile Val Val Val 85 90 95 Lys Ala Val Ser CysVal Met Val Ala His His Gly Glu Ala Val Gln 100 105 110 Thr Thr Asn AlaAla Thr Val Val Glu Asn Leu Val Asp Tyr Ile Asn 115 120 125 Asn Pro Arg130

What is claimed is:
 1. An isolated Aspergillus fumigatus profilin protein, wherein the protein 1) comprises a sequence having greater than 95% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm, and 2) binds monomeric actin.
 2. An isolated protein of claim 1, wherein the protein has a sequence that has greater than 98% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm.
 3. The isolated protein of claim 1, wherein the protein specifically binds to polyclonal antibodies generated against a protein comprising SEQ ID NO:2.
 4. An isolated protein wherein the protein comprises the amino acid sequence of SEQ ID NO:2. 